Triterpenoid derivatives

ABSTRACT

The present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in therapy. Preferably, the compound may be used for treating a patient suffering from leukaemia, cancer or other proliferative disorder. A further embodiment relates to the use of a compound of formula (I) in an assay for detecting the phosphorylation and acetylation state of cellular substrates. The present invention also relates to novel compounds of formula (Ia).

RELATED APPLICATIONS

This application is a 35 U.S.C. §371 filing of International ApplicationNumber PCT/GB01/02313 which was filed 23 May 2001, which claims priorityto Great Britain Publication 0012526.0, filed 23 May 2000 in GreatBritain. The contents of the aforementioned application are herebyincorporated by reference.

The present invention relates to the therapeutic use and the biologicalactivity of triterpenoid derivatives. The invention further relates tonovel triterpenoid derivatives.

To date, the prior art has primarily focussed on compounds that arecapable of regulating the cell cycle by virtue of inhibiting cyclindependent kinases (CDKs). Examples of such compounds includebutyrolactone I, flavopiridol, bohemin, olomoucine, roscovitine,purvanalol and indarubicine.

There is considerable support in the literature for the hypothesis thatCDKs and their regulatory proteins play a significant role in thedevelopment of human tumours. Thus, in many tumours a temporal abnormalexpression or activity of CDKs has been observed, together with a majorderegulation of protein inhibitors (mutations, deletions). This resultsin the activation of CDKs and consequently in defective regulation ofthe G1/S transition. Unlike normal cells, tumour cells do not arrest inG1, and since they become independent of growth factors, they pass theG1 restriction point and enter the S phase very rapidly.

In contrast to the prior art, the present invention relates to compoundswhich are anti-proliferative, but which are believed to operate via amechanism other than CDK inhibition.

The G₁/S transition of the mammalian cell cycle is tightly regulated bythe retinoblastoma protein (pRb). Retinoblastoma gene mutations ordeletions predispose individuals to familiar retinoblastoma and othertypes of cancers. The pRb protein is a docking protein, which inhypophosphorylated form has the capacity to bind and thus to inactivateS-phase transcription factors such as DP-1 and E2F. However, followingphosphorylation by G₁/S cyclin-dependent kiases (CDKs) (CDK4/cyclinD1–D3, CDK6/cyclin D1–D3, CDK2/cyclin A), hyperphosphorylated pRbreleases the transcription factors and S phase is initiated. Within theS phase, the pRb protein phosphorylation is maintained by the activityof CDK2/cyclin E complexes. Thus, hyperphosphorylation of the pRbprotein plays a key role in the molecular pathology of cancer cells withaltered CDK activity.

The present invention relates to the use of triterpenoid compoundsderived from the natural products betulin and betilinic acid (BA) asshown in formula (A). The compounds of the present invention arereferred to hereinafter as betulinines.

With regard to their biological and therapeutic activity, the compoundsdisclosed herein are believed to be of specific benefit in the treatmentof proliferative diseases such as cancers and leukaemias.

Several of the compounds suitable for use in the present invention arealready known in the art, for example those disclosed in Ber. Dtsch.Chem. Ges. 55, 2332 (1922), Schluze, H. et al; Acta Chem. Scand., B 29,139 (1975), Suokas E. et al; Collect Czech. Chem. Commun. 56, 2936(1991), Sejbal J. et al; Collect. Czech. Chem. Commun. 64, 329 (1999),Klinotová et al; Indian. J. Chem., Sect. B 34, 624 (1995), Dinda B. etal, Chem. Listy 91, 1005 (1997), {hacek over (S)}arek J. et al. However,these disclosures do not include any indication as to possiblebiological activity of such compounds.

A first aspect of the present invention relates to the use of a compoundof formula I, or a pharmaceutically acceptable salt thereof, in therapy,

wherein:

-   -   X¹ is C═O, C═NOR^(1a), CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal,        CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I,        F;    -   X³ is C═O, CHOR^(1b), CHOCOR^(1b), or X³ and R⁸ together are        CHOCOCH₂ and form a spirolactone;    -   R¹⁻⁵ are each independently H or lower alkyl;    -   R⁶ is H or absent if “a” is a double bond;    -   R⁷ is H, COOR^(1c), YOCOR^(1c), COOYOCOR^(1e), YCOOR^(1e);    -   R⁸ is H COOR^(1d), YCOOR^(1d), YCOOR¹⁰, YCOHal, COOYOCOR^(1d),        CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d) or R⁷ and R⁸ together        are ═CH₂ or CH₂OCOCH₂;    -   R⁹ is an OH-substituted alkyl group, an ether group or a cyclic        ether;    -   R¹⁰ is lower alkyl substituted by Hal    -   “a” is a double bond or a single bond    -   and wherein Y═(CH₂)n        -   n=0 to 5;        -   R^(1a-1d) are the same or different groups of R¹.

In a preferred aspect, the invention relates to the use of a compound offormula I, or a pharmaceutically acceptable salt thereof, for treating apatient suffering from leukaemia, cancer or other proliferativedisorder.

A second aspect of the present invention relates to novel betulinines ofstructural formula Ia, or pharmaceutically acceptable salts, thereof;

wherein:

-   -   X¹ is C═O, C═NOR^(1a), CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal,        CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I,        F;    -   X³ is C═O, CHOR^(1b), CHOCOR^(1b), or X³ and X⁸ together are        CHOCOCH₂ and form a spirolactone;    -   R¹⁻⁵ are each independently H or lower alkyl;    -   R⁶ is H or absent if “a” is a double bond;    -   R⁷ is H, COOR^(1c), YOCOR^(1c), COOYOCOR^(1e), YCOOR^(1e);    -   R⁸ is H, COOR^(1d), YCOOR^(1d), YCOOR¹⁰, YCOHal, COOYOCOR^(1d),        CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d) or R⁷ and R⁸ together        are ═CH₂ or CH₂OCOCH₂;    -   R⁹ is an OH-substituted alkyl group, an ether group or a cyclic        ether;    -   R¹⁰ is lower alkyl substituted by Hal    -   “a” is a double bond or a single bond    -   and wherein Y═(CH₂)n        -   n=0 to 5;        -   R^(1a-1d) are the same or different groups of R¹            with the proviso that    -   (i) when X¹ is CHOAc, X³ is C═O, “a” is single bond, R¹⁻⁵ are        Me, R⁶ is H;        -   when R⁷ is CH₂OAc, R⁸ is other than COOH, CH₂COCOPr^(i),            COCOCOPr^(i), CH₂COOH, or CH₂CH₂CO^(i)Pr;        -   when R⁷ is CO₂Me, R⁸ is other than CH₂CH₂COCH(CH₃)₂;        -   when R⁷is H, R⁸ is other than H, CH₂COOMe, CH₂COOH or            CH₂CH₂COPr^(i); and    -   (ii) when X¹ is CHOH, X³ is C═O, “a” is single bond, R¹⁻⁵ are        Me, and R⁶ is H,        -   when R⁷ is H, R⁸ is other than H, CH₂COOH, CH₂CH₂COCH(CH₃)₂            or CH₂COOMe;        -   when R⁷ is CH₂OAc, R⁸ is other than CH₂COOH;        -   or a pharmaceutically acceptable salt thereof.

As used herein, the term lower alkyl means a linear or branched chainalkyl group containing from 1 to 6 carbon atoms, including methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Within the options provided for the groups X¹, X³. and R¹⁻⁸ of formulaI, the following options are preferred;

Preferably,

-   -   X¹ is CHOR^(1a), CHOCOR^(1a) or CHOCOY-Hal; and    -   R⁸ is H, COOR^(1d), YCOOR^(1d), COOYOCOR^(1d), CH₂OR^(1d),        CH₂COCOR^(1d) or COCOCOR^(1d).

In a more preferred embodiment, R²⁻⁵ are all methyl and R¹ is as definedbelow for the relevant group R^(1a-1d);

-   -   X¹ is —CHOCOCH₂Cl; or        -   —CHOR^(1a) or CHOCOR^(1a), wherein R^(1a) is H and methyl            respectively;    -   X³ is C═O, CHOH or CHOAc;    -   R⁷ is H, COOH, COOMe, CH₂OAc, COOYOCOR^(1e) or YCOOR^(1e) where    -   Y is CH₂ and R^(1e) is C₁₋₄ alkyl;    -   R⁸ is —COOR^(1d), wherein R^(1d) is H or methyl;        -   —YCOOR^(1d), wherein Y is CH₂ and R^(1d) is H, methyl or            ethyl;        -   —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is C₁₋₄ alkyl;        -   —CH₂OR^(1d), wherein R^(1d) is C₁₋₄ alkyl;        -   —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄            alkyl.

Of the preferred definitions provided above, it is preferable that;

-   -   R⁸ is —COOYOCOR^(1d), wherein Y is CH₂ and R^(1d) is methyl or        butyl;        -   —CH₂OR^(1d), wherein R^(1d) is methyl or ethyl;        -   —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is propyl;            and    -   R⁷ is COOYOCOR^(1e) or YCOOR^(1e) where Y is CH₂ and R^(1e) is        methyl or butyl.

In a preferred embodiment, “a” is a single bond and R⁶ is H.

In a more preferred embodiment of the first aspect of the invention, thecompounds of use are selected from those shown in Table 1 below.

TABLE 1 No. X¹ R¹ X³ a R^(2–5) R⁶ R⁷ R⁸ I.1 CHOAc CH₃ C═O single Me HCH₂OAc CH₂COCOPr¹ I.2 CHOAc CH₃ C═O single Me H CH₂OAc COCOCOPr¹ I.3CHOAc CH₃ C═O single Me H CH₂OAc COOH I.4 CHOAc CH₃ C═O single Me HCH₂OAc COOMe I.5 CHOAc CH₃ C═O single Me H H H I.6 CHOAc CH₃ C═O singleMe H H CH₂COOH I.7 CHOAc CH₃ C═O single Me H H CH₂OEt I.8 CHOAc CH₃ CHOHsingle Me H CH₂OAc COOH I.9 CHOAc CH₃ C═O double Me absent CH₂OAc COOMeI.10 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ I.11 CHOAc CH₃CHOAc single Me H CH₂OAc COOMe I.12 CHOAc CH₃ C═O single Me H COOMeCH₂COCOPr¹ I.13 CHOAc CH₃ C═O single Me H COOMe COOH I.14 CHOAc CH₃ C═Osingle Me H COOMe CH₂COOH I.15 CHOAc CH₃ CHOH single Me H CH₂OAcCOOCH₂OCOBu^(t) I.16 CHOAc CH₃ CHOH single Me H CH₂OAc COOMe I.17 CHOAcCH₃ C═O single Me H CH₂OAc CH₂COOH I.18 CHOAc CH₃ C═O single Me H CH₂OAcCOOCH₂OCOBu^(t) I.19 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOMe I.20CHOAc CH₃ C═O double Me absent CH₂OAc COOCH₂OCOBu^(t) I.21 CHOAc CH₃ C═Odouble Me absent CH₂OAc COOCH₂OCOMe I.22 CHOAc CH₃ C═O double Me absentCH₂OAc COOH I.24 CHOAc CH₃ C═O single Me H R⁷ and R⁸ together: ═CH₂ I.26CHOH H C═O single Me H CH₂OAc COOH I.27 CHOH H C═O double Me absentCH₂OAc COOMe I.28 CHOAc CH₃ C═O single Me H COOCH₂OCOMe CH₂COCOPr¹ I.29CHOAc CH₃ C═O single Me H COOCH₂OCOBu^(t) CH₂COCOPr¹ I.30 CHOAc CH₃ C═Osingle Me H from R⁷ to R⁸: CH₂OC(O)CH₂ I.31 CHOAc CH₃ CHOAc single Me HCH₂OAc COOH I.32 CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) I.33CHOAc CH₃ CHOH single Me H CH₂OAc COOMe I.34 CHOAc CH₃ CHOAc single Me HCH₂OAc COOMe I.35 CHOAc CH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) I.36CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) I.37 CHOAc CH₃ C═Osingle Me H CH₂OAc COOMe I.38 CHOAc CH₃ C═O single Me H H CH₂COOMe I.39CHOAc CH₃ C═O single Me H COOMe CH₂COOMe I.40 CHOAc CH₃ C═O single Me HCOOCH₂OCOBu^(t) CH₂COOMe I.41 CHOAc CH₃ C═O single Me H COOCH₂OCOMeCH₂COOMe I.42 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ I.43CHOAc CH₃ CHOH single Me H H H I.44 CHOCOCH₂Cl CH₃ C═O single Me HCH₂OAc COOH I.45 CHOAc CH₃ CHOH single Me H CH₂OAc COOH I.46 CHOAc CH₃CHOAc single Me H CH₂OAc COOH I.47 CHOAc CH₃ CHOAc single Me H R⁷ and R⁸together: ═CH₂ I.48 CHOAc CH₃ CHOAc single Me H H H I.49 CHOH CH₃ C═Osingle Me H H CH₂COOH I.50 CHOH CH₃ C═O single Me H H CH₂COOMe I.51 CHOHCH₃ CHOH single Me H H CH₂COOMe I.52 CHOAc CH₃ CHOH single Me H HCH₂COOMe I.53 CHOH CH₃ CHOH single Me H CH₂COOMe H I.54 CHOAc CH₃ CHOAcsingle Me H CH₂COOMe H I.55 CHOAc CH₃ {circle around (1)} single Me H H{circle around (1)} I.56 CHOH CH₃ {circle around (1)} single Me H H{circle around (1)} I.57 CHOH CH₃ C═O single Me H CH₂OAc COOH I.58 CHOAcCH₃ C═O single Me H CH₂OAc COF where: {circle around (1)} means cyclicfive-membered spirolactone from X³ to R⁸: OCOCH₂

In a most preferred embodiment, the compound is selected from;

In respect of the second aspect of the invention, the preferredembodiments regarding the compounds are identical to those given abovefor the first aspect with application of the proviso of formula Ia.

The most preferred compounds of the second aspect are those in Table 1abelow.

TABLE 1a No. X¹ R¹ X³ a R^(2–5) R⁶ R⁷ R⁸ I.4 CHOAc CH₃ C═O single Me HCH₂OAc COOMe I.7 CHOAc CH₃ C═O single Me H H CH₂OEt I.8 CHOAc CH₃ CHOHsingle Me H CH₂OAc COOH I.9 CHOAc CH₃ C═O double Me absent CH₂OAc COOMeI.10 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ I.11 CHOAc CH₃CHOAc single Me H CH₂OAc COOMe I.12 CHOAc CH₃ C═O single Me H COOMeCH₂COCOPr¹ I.13 CHOAc CH₃ C═O single Me H COOMe COOH I.14 CHOAc CH₃ C═Osingle Me H COOMe CH₂COOH I.15 CHOAc CH₃ CHOH single Me H CH₂OAcCOOCH₂OCOBu^(t) I.16 CHOAc CH₃ CHOH single Me H CH₂OAc COOMe I.18 CHOAcCH₃ C═O single Me H CH₂OAc COOCH₂OCOBu^(t) I.19 CHOAc CH₃ C═O single MeH CH₂OAc COOCH₂OCOMe I.20 CHOAc CH₃ C═O double Me absent CH₂OAcCOOCH₂OCOBu^(t) I.21 CHOAc CH₃ C═O double Me absent CH₂OAc COOCH₂OCOMeI.22 CHOAc CH₃ C═O double Me absent CH₂OAc COOH I.24 CHOAc CH₃ C═Osingle Me H R⁷ and R⁸ together: ═CH₂ I.26 CHOH H C═O single Me H CH₂OAcCOOH I.27 CHOH H C═O double Me absent CH₂OAc COOMe I.28 CHOAc CH₃ C═Osingle Me H COOCH₂OCOMe CH₂COCOPr¹ I.29 CHOAc CH₃ C═O single Me HCOOCH₂OCOBu^(t) CH₂COCOPr¹ I.30 CHOAc CH₃ C═O single Me H from R⁷ to R⁸:CH₂OC(O)CH₂ I.31 CHOAc CH₃ CHOAc single Me H CH₂OAc COOH I.32 CHOAc CH₃CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) I.33 CHOAc CH₃ CHOH single Me HCH₂OAc COOMe I.34 CHOAc CH₃ CHOAc single Me H CH₂OAc COOMe I.35 CHOAcCH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) I.36 CHOAc CH₃ CHOAc singleMe H CH₂OAc COOCH₂OCOBu^(t) I.37 CHOAc CH₃ C═O single Me H CH₂OAc COOMeI.39 CHOAc CH₃ C═O single Me H COOMe CH₂COOMe I.40 CHOAc CH₃ C═O singleMe H COOCH₂OCOBu^(t) CH₂COOMe I.41 CHOAc CH₃ C═O single Me H COOCH₂OCOMeCH₂COOMe I.42 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ I.43CHOAc CH₃ CHOH single Me H H H I.44 CHOCOCH₂Cl CH₃ C═O single Me HCH₂OAc COOH I.45 CHOAc CH₃ CHOH single Me H CH₂OAc COOH I.46 CHOAc CH₃CHOAc single Me H CH₂OAc COOH I.47 CHOAc CH₃ CHOAc single Me H R⁷ and R⁸together: ═CH₂ I.48 CHOAc CH₃ CHOAc single Me H H H I.51 CHOH CH₃ CHOHsingle Me H H CH₂COOMe I.52 CHOAc CH₃ CHOH single Me H H CH₂COOMe I.53CHOH CH₃ CHOH single Me H CH₂COOMe H I.54 CHOAc CH₃ CHOAc single Me HCH₂COOMe H I.55 CHOAc CH₃ {circle around (1)} single Me H H {circlearound (1)} I.56 CHOH CH₃ {circle around (1)} single Me H H {circlearound (1)} I.57 CHOH CH₃ C═O single Me H CH₂OAc COOH I.58 CHOAc CH₃ C═Osingle Me H CH₂OAc COF where: {circle around (1)} means cyclicfive-membered spirolactone from X³ to R⁸: OCOCH₂

In respect of the invention as a whole, it is preferable that theproliferative disorder is cancer or leukaemia. In one embodiment, thecancer or leukaemia is p53, hormone and multidrug resistanceindependent. In another embodiment, the cancer or leukaemia isindependent of Rb status.

More specifically, the present invention relates to a method of treatingpatients suffering from cancer by administering therapeuticallyeffective amounts of a compound of formula I or pharmaceuticallyacceptable salts or esters thereof.

Without wishing to be bound by theory, preliminary studies suggest thatrather than influencing the activity of cyclin dependent kinases, thecompounds of the present invention appear to operate via an alternativemechanism. In particular, it is believed that the betulinines of thepresent invention may inhibit cell proliferation and induce cancer celldeath in a manner which involves mainly post-translationalmodifications, namely the phosphorylation, of a key regulatory proteininvolved in cellular proliferation. More specifically, it is believedthat the betulinines of the invention effect a change in thephosphorylation state of the Rb protein. Such a mechanism may beadvantageous as it is thought that the compounds of the presentinvention may be capable of inhibiting cell proliferation inproliferating tumour tissue, but not in healthy tissue.

Thus, in a further embodiment the present invention relates to a methodof treating a cancerous or leukaemic proliferative disease througheffecting a change in the pRb protein phosphorylation state by theadministration of a therapeutically effective amount of a compound offormula I or pharmaceutically acceptable salts or esters thereof.

The compounds of the present invention are also capable of inducingapoptosis (programmed cell death) in proliferative cells. Thus, in anadditional embodiment, the present invention relates to a method ofinducing cell death in proliferative cells comprising administering atherapeutically effective amount of a compound of formula I orpharmaceutically acceptable salts or esters thereof.

A further aspect of the present invention relates to use of betulininesof formula I as research chemicals and as compounds for clinical and/orlaboratory diagnostics. More particularly, the invention relates to theuse of betulinines as research chemicals for studying thephosphorylation/de-phosphorylation processes of cellular substrates,cellular proliferation, purification of target molecules, and/or cellcycle studies.

The present invention therefore further relates to the use of a compoundof formula I in the preparation of a medicament for use in the treatmentof a proliferative disease.

As used herein the phrase “preparation of a medicament” includes the useof a compound of formula I directly as the medicament in addition to itsuse in a screening programme for the identification of furtheranti-proliferative agents or in any stage of the manufacture of such amedicament

Such a screening programme may for example include an assay fordetermining the phosphorylation state of cellular substrates anddetermining whether a candidate substance is capable of mimicking theactivity of a betulinine of formula I.

Thus, in a further embodiment, the invention relates to the use of acompound of formula I or a pharmaceutically acceptable salt, crystalform, complex, hydrate, or hydrolysable ester thereof, in an assay fordetermining the phosphorylation state of cellular substrates, andoptionally in the identification of candidate compounds that act in asimilar manner.

Preferably, the cellular substrate, the phosphorylation state of whichis being assayed is Rb protein.

The compounds of the fir&t and second aspects of the present inventioncan be present as salts or esters, in particular pharmaceuticallyacceptable salts or esters.

Pharmaceutically acceptable salts of the product of the inventioninclude suitable acid addition or base salts thereof A review ofsuitable pharmaceutical salts may be found in Berge et al, J Pharm Sci,66, 1–19 (1977). Salts are formed, for example with strong inorganicacids such as mineral acids, e.g. sulphuric acid, phosphoric acid orhydrohalic acids; with strong organic carboxylic acids, such asalkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted orsubstituted (e.g., by halogen), such as acetic acid; with saturated orunsaturated dicarboxylic acids, for example oxalic, malonic, succinic,maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylicacids, for example ascorbic, glycolic, lactic, malic, tartaric or citricacid; with amino acids, for example aspartic or glutamic acid; withbenzoic acid; or with organic sulfonic acids, such as (C₁–C₄)alkyl- oraryl-sulfonic acids which are unsubstituted or substituted (for example,by a halogen) such as methane- or p-toluene sulfonic acid.

Esters are formed either using organic acids or alcohols/hydroxides,depending on the functional group being esterified. Organic acidsinclude carboxylic acids, such as alkanecarboxylic acids of 1 to 12carbon atoms which are unsubstituted or substituted (e.g., by halogen),such as acetic acid; with saturated or unsaturated dicarboxylic acid,for example oxalic, malonic, succinic, maleic, fumaric, phthalic ortetraphthalic; with hydroxycarboxylic acids, for example ascorbic,glycolic, lactic, malic, tartaric or citric acid; with amino acids, forexample aspartic or glutamic acid; with benzoic acid; or with organicsulfonic acids, such as (C₁–C₄)-alkyl- or aryl-sulfonic acids which areunsubstituted or substituted (for example, by a halogen) such asmethane- or p-toluene sulfonic acid. Suitable hydroxides includeinorganic hydroxides, such as sodium hydroxide, potassium hydroxide,calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcoholsof 1–12 carbon atoms which may be unsubstituted or substituted, e.g. bya halogen).

In all aspects of the present invention previously discussed, theinvention includes, where appropriate all enantiomers and tautomers ofcompounds of formula I or Ia. The man skilled in the art will recognisecompounds that possess optical properties (one or more chiral carbonatoms) or tautomeric characteristics. The corresponding enantiomersand/or tautomers may be isolated/prepared by methods known in the art.

The invention furthermore relates to the compounds of, or of use, in thepresent invention in their various crystalline forms, polymorphic formsand (an)hydrous forms. It is well established within the pharmaceuticalindustry that chemical compounds may be isolated in any of such forms byslightly varying the method of purification and or isolation from thesolvents used in the synthetic preparation of such compounds.

The invention further includes the compounds of, or of use, in thepresent invention in prodrug form. Such prodrugs are generally compoundsof formula I or Ia wherein one or more appropriate groups have beenmodified such that the modification is reversed upon administration to ahuman or mammalian subject. Such reversion is usually performed by anenzyme naturally present in such subject, though it is possible for asecond agent to be administered together with such a prodrug in order toperform the reversion in vivo. Examples of such modifications includeesters (for example, any of those described above), wherein thereversion may be carried out be an esterase etc. Other such systems willbe well known to those skilled in the art.

The present invention also encompasses pharmaceutical compositionscomprising the compounds of the invention. In this regard, and inparticular for human therapy, even though the compounds of the presentinvention (including their pharmaceutically acceptable salts, esters andpharmaceutically acceptable solvates) can be administered alone, theywill generally be administered in admixture with a pharmaceuticalcarrier, excipient or diluent selected with regard to the intended routeof administration and standard pharmaceutical practice.

Thus, the present invention also relates to pharmaceutical compositionscomprising betulinines or pharmaceutically acceptable salts or estersthereof, together with at least one pharmaceutically acceptableexcipient, diluent or carrier.

By way of example, in the pharmaceutical compositions of the presentinvention, the compounds of the invention may be admixed with anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),and/or solubilising agent(s). Examples of such suitable excipients forthe various different forms of pharmaceutical compositions describedherein may be found in the “Handbook of Pharmaceutical Excipients”,2^(nd) Edition, (1994), Edited by A Wade and P J Weller.

The pharmaceutical compositions of the present invention may be adaptedfor oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal,intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal,intravenous, nasal, buccal or sublingual routes of administration.

For oral administration, particular use is made of compressed tablets,pills, tablets, gellules, drops, and capsules. Preferably, thesecompositions contain from 1 to 250 mg and more preferably from 10–100mg, of active ingredient per dose.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally or intramuscularly, andwhich are prepared from sterile or sterilisable solutions. Thepharmaceutical compositions of the present invention may also be in formof suppositories, pessaries, suspensions, emulsions, lotions, ointments,creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredient can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredient can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

Injectable forms may contain between 10–1000 mg, preferably between10–250 mg, of active ingredient per dose.

Compositions may be formulated in unit dosage form, i.e., in the form ofdiscrete portions containing a unit dose, or a multiple or sub-unit of aunit dose.

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will vary with the age, weight and response ofthe particular patient The dosages disclosed herein are exemplary of theaverage case. There can of course be individual instances where higheror lower dosage ranges are merited, and such are within the scope ofthis invention.

In an exemplary embodiment, one or more doses of 10 to 150 mg/day willbe administered to the patient for the treatment of malignancy.

The invention further relates to methods of chemical synthesis of theabove described compounds.

In one embodiment, the invention relates to a process for preparingcompounds of formula I as defined above, comprising:

-   -   (i) oxidising a compound of formula Ib to a compound of formula        Ic;    -   (ii) reducing said compound of formula Ic to form a compound of        formula Id, and optionally    -   (iii) converting said compound of formula Id to a compound of        formula I wherein “a” is a double bond.

In a preferred embodiment, the compound of formula Ib is oxidised to Icby treating sequentially with selenium dioxide, peroxyacetic acid andruthenium tetroxide.

In a more preferred embodiment, the reduction of Ic to Id is astereoselective reduction.

Even more preferably, the compound of formula Ic is reduced to Id bytreating with sodium borohydride in the presence of a cerium (III) salt.

In a preferred aspect, step (iii) comprises esterifying a compound offormula Id wherein R^(1d) is H, oxidising and dehydrogenating.

The preparation of the compounds of the present invention will bediscussed in greater detail below, with specific reference to thepreferred embodiments. The man skilled in the relevant art would be ableto prepare other compounds of the invention by selection of theappropriate reagents.

The following scheme illustrates the synthesis of compounds of formula Iwhere X¹ is CHOAc, X² is CH₂, X³ is C═O, R¹⁻⁵ are methyl, R⁶ is H orabsent (when “a” is a double bond), R⁷ is CH₂OAc, and R⁸ is COOH,COOCH₂OCOMe, or COOCH₂OCOC(CH₃)₃.

Conditions: a, Acetylation with acetic anhydride in the presence of base(e.g. pyridine); b, isomerisation of double bond by treatment withhydrogen bromide in acetic acid; c, oxidation (e.g. with sodiumdichromate); d, oxidation (e.g with selenium dioxide); e, oxidation(e.g. with peroxyacetic acid); f, fission of double bond (e.g. withruthenium tetroxide); g, stereoselective reduction (e.g. with sodiumborohydride in the presence of a cerium(III) salt); h, esterificationwith chloromethyl pivalate (POM-Cl) in the presence of base (e.g.1,8-diazabicyclo[5,4,0]undec-7-ene (DBU)); i, esterification withbromomethyl acetate (AcM-Br) in the presence of base (e.g. DBU); j,dehydrogenation (e.g. with selenium dioxide); k, hydrolysis (e.g. withbis(tributyltin)oxide).

The scheme below illustrates the synthesis of compound I.58 of formula Iwhere X¹ is CHOAc, X³ is C═O, R¹–R⁵ are methyls, R⁶ is H, R⁷ is CH₂OAc,R⁸ is COF.

Conditions: 1, reaction with diethylaminosulphur trifluoride.

The present invention is also described with reference to theaccompanying figures wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an electron micrograph of a normal, untreated A549 lungcancer cell attached to coverslip with a number of well formedpseudopods and fine structure of cytoplasmatic membrane.

FIG. 1B shows an electron micrograph of betulinic acid treated A549 lungcancer cells. The left cell is attached to the coverslip and exhibitsnormal morphology with fine cytoplasmatic membrane structure, while theright cell already shows cytoplasmatic membrane blebbing, an early signof apoptosis.

FIG. 1C shows an electron micrograph of I.3 treated A549 lung cancercells. This illustrates a typical apoptotic tumour cell with extensivecytoplasmatic membrane blebbing, detachment and formation of apoptoticbodies.

FIG. 1D is a magnification of FIG. 1C, showing details of apoptotic bodyformation in I.3 treated cells.

FIG. 2 shows the anticancer activity of betulinine I.3 inxenotrasplantated human SK-N-AS neuroblastoma

FIG. 3 shows the results of SDS-PAGE electrophoresis of total cellularproteins. In particular, the gel shows that betulinine I.3, but notbetulinic acid (BA) induces rapid dephosphorylation of Rb protein.

FIG. 4A shows a cell cycle analysis of cells treated with I.3 andpaclitaxel as a control.

FIG. 4B shows the induction of apoptosis in cells treated with I.3 orpaclitaxel as a control.

More detailed reference to the above figures may be found in theaccompanying examples.

This invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES

General

The chemical shift values (δ-scale, ppm) and coupling constants (J, Hz)in the ¹H and ¹³C NMR spectra were obtained using a Varian UNITY-INOVA400 FT spectrometer (¹H at 400 MHz and ¹³C at 100.6 MHz) indeuterochloroform with tetramethylsilane (for ¹H NMR data δ=0 ppm) as aninternal standard. For the ¹³C NMR data δ(CDCl₃)=77.00 ppm. The valuefor a multiplet, either defined (doublet (d), triplet (t), quartet (q),septet (sept) or not (m) at the approximate mid point is given unless arange is quoted (s=singlet, b=broad)).

Electron impact mass spectra (EIMS) were measured on an INCOS 50instrument ionising electron energy 75 eV, ion source temperature 150°C. EIMS was used to determine molecular weights, M⁺ corresponding to themolecular ion.

Ether is diethylether. THF and dioxane were dried over sodium. Aceticacid was purified before use by chromium trioxide treatment anddistillation. Reactions were run at room temperature unless otherwisestated. The reaction progress was monitored by thin layer chromatography(TLC) on silicagel 60 G (Merck, detection by spraying with 10% sulphuricacid and heating). The work-up procedure involves dilution withspecified solvent (otherwise the organic reaction solvent), extractionwith water and then brine or sodium hydrogencarbonate, drying overanhydrous magnesium sulphate, and evaporation under vacuum to give aresidue.

Example 1 Lup-20(29)-ene-3β,28-diyl diacetate

Crude betuline (500 g) was dissolved in a mixture of 250 ml pyridine and250 ml acetic anhydride. The mixture was then refluxed for half an hour.After cooling, the resulting crystals were filtered off and washed withacetic acid, ethanol and water. A solution of crudelup-20(29)-ene-3β,28-diyl diacetate (400 g) in chloroform was filteredthrough a column of alumina, and the column was washed with chloroform.The filtrate was then evaporated under reduced pressure. The residue wascrystallized from chloroform/methanol to obtain 250 g of the titlecompound which according to TLC contained traces of lupeol acetate.After re-crystallization from chloroform/methanol, the yield of purecompound was 239 g, mp. 222–223° C., [α]_(D)+22° (c 0.4; CHCl₃).[Schulze H., Pieroh K: Ber. Dtsch Chem. Ges. 55,2332 (1922)].

The ¹H NMR spectrum of the title compound is as follows: 0.84 s, 0.84 s,0.85 s, 0.97 s, 1.03 s, 1.68, 6×3H (6×CH₃); 2.04 s, 3 H, 2.07 s, 3 H(2×OAc); 2.44 ddd, 1 H (J′=11.4, J″=10.9, J′″=0.7, H-19); 3.85 d, 1 H(J=11.1, H-28a); 4.25 dd, 1 H, (J′=11.1, J″=1.4, H-28b); 4.47 m, 1 H(H-3α); 4.59 m, 1 H (ΣJ=3.4, H-29E); 4.69 m, 1 H (ΣJ=2.1, H-29Z).

Example 2 Lup-18-ene-3β,28-diyl diacetate

A solution of hydrogen bromide in acetic acid (38%, 1.4 l) was added toa solution of lup-20(29)-ene-3β,28-diyl diacetate (100 g, 190 mmol) in amixture of benzene, acetic acid and acetic anhydride (11:0.51:50 ml).The reaction mixture was refluxed until the reaction was completed (TLCwas developed in hexane/ether mixture). After cooling, the reactionmixture was poured into ice cold water (3:1) and extracted with benzene(3×0.5 l). The combined organic phases were washed with NaHCO₃ aqueoussolution, NaHSO₃ solution and water and dried over magnesium sulphate.Usual working up procedure gave 90 g of dark brown residue. The drypowder was extracted in a Soxhlet extractor with acetone until it turnedwhite. After drying in the air, the product was crystallized frombutanone. The yield of the title compound was 74 g (74%), mp. 215–216°C., [α]_(D)+15° (c 0.45; CHCl₃). [Suokas E., Hase T.: Acta Chem. Scand.,B 29, 139 (1975)].

The ¹H NMR spectrum of the title compound is as follows: 0.84 s, 0.85 s,0.89 s, 0.90 s, 0.91 d, 3 H (J=6.8), 0.99 d, 3 H (J=6.8), 1.06 s, 7×3H(7×CH₃); 2.04 s, 3 H, 2.05 s, 3 H (2×OAc); 2.25 m, 2 H (ΣJ˜15); 2.43 m,1 H (ΣJ˜15); 3.14 sept., 1 H (J=7, H-20); 3.98 d, 1 H (J=10.8, H-28a);4.03 d, 1 H (J=10.8, H-28b); 4.49 m, 1 H (H-3α).

Example 3 21-oxo-lup-18-ene-3β,28-diyl diacetate

Lup-18-ene-3β,28-diyl diacetate (50 g; 95 mmol), sodium dichromate (22.5g; 75.5 mmol) and sodium acetate (5 g) were dissolved in a mixture ofbenzene and acetic acid (0.7 l, 0.3 l). The reaction mixture was allowedto stand until the reaction was completed (TLC was developed inhexane/ether). After dilution with an excess of water, the mixture wasextracted with benzene (3×300 ml). After usual working up procedure thetitle compound was obtained (45 g, 87%) as a pale-yellow crystaline foamwhich was used in the next step without further purification (seeExample 4). Pure title compound has m.p. 205–206° C., [α]_(D)−35° (c0.49; CHCl₃). Another way to the title compound is described in SejbalJ., Klinot J., Bude{hacek over (s)}ínský M., Protiva J.: Collect. CzechChem. Commun. 56,2936 (1991).

The ¹H NMR spectrum of the title compound is as follows:

0.85 s, 0.86 s, 0.93 s, 0.94 s, 1.16 s, 1.17 d (J=7.1), 1.21 d (J=7.1),7×3 H (7×CH₃); 2.00 s, 3 H, 2.05 s, 3 H (2×OAc); 2.39 d, 1 H (J=18.5,H-22); 2.87 dd, 1 H (J′=11.9, J″=4.1, H-13β); 3.18 sept, 1 H (J=6.6,H-20); 4.06 d, 1 H (J=10.9, H-28a); 4.34 d, 1 H (J=10.9, H-28b); 4.49 m,1 H (J˜7, H-3α).

The following compounds were prepared by the above-mentioned procedure:

-   -   (pivaloyloxy)methyl        3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oate    -   acetoxymethyl        3β,28diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oate

Example 4 21,22-dioxolup-18-ene-3β,28-diyl diacetate

A solution of crude 21-oxolup-18-ene-3β,28-diyl diacetate (40 g; 74mmol; containing about 85% of 21-oxo-lup-18-ene-3β,28-diyl diacetate)and selenium dioxide (160 g; 1.44 mol) in a mixture of dioxane (0.8 l)and acetic acid (0.4 l) was refluxed until the reaction was completed(TLC was developed in benzene/ether).

After cooling, the precipitated selenium was removed by filtration andthe filtrate was slowly poured into a vigorously stirred excess ofwater. The red-orange precipitate was filtered off under reducedpressure, carefully washed with water and dried in the air. Dry crude21,22dioxo-lup-18-ene-3β,28-diyl diacetate was dissolved in chloroformand the solution was filtered through a column of alumina, the columnwas then washed with chloroform, and the filtrate was evaporated underreduced pressure. The residue was crystallized from methyl acetate togive 28.9 g (82%) of the title compound as pale-orange needles, mp.267–270° C., [α]_(D)−127° (c 0.32; CHCl₃). Another way to the titlecompound is described in Klinotová E., Cermáková J., Rejzek M., KrecekV., Sejbal J., Ol{hacek over (s)}ovský P., Klinot J.: Collect. CzechChem. Commun. 64, 329 (1999).

The ¹H NMR spectrum of the title compound is as follows: 0.85 s, 0.86 s,0.94 s, 0.97 s, 1.18 s, 1.24 d (J=7.2), 1.26 d (J=7.2), 7×3 H (7×CH₃);1.93 s, 3 H, 2.06 s, 3 H (2×OAc); 3.12 dd, 1 H (J′=12.5, J″=3.8, H-13β),3.36 sept., 1 H (J=7.0, H-20); 4.02 d, 1 H (J=11.1, H-28a); 4.49 dd, 1 H(J′=10.2, J″=6.0, H-3α); 4.84 d, 1 H (J=11.1, H-28b).

The following compounds were prepared by the above-mentioned procedure:

methyl 3β-acetoxy-21,22dioxolup-18-en-28-oate

acetoxymethyl 3β-acetoxy-21,22-dioxolup-18-en-28-oate

(pivaloyloxy)methyl 3β-acetoxy-21,22-dioxolup-18-en-28-oateacetoxymethyl3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-12-en-22-oate

(pivaloyloxy)methyl3β,28diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-12-en-22-oate

3β-hydroxy-30-oxolup-20(29)-en-28-oic acid [Dinda B., Hajra A. K., DasS. K., Chel G., Chakraborty R., Ranu B. C.: Indian. J. Chem., Sect. B34, 624 (1995)].

acetoxymethyl 3β-hydroxy-30-oxolup-20(29)-en-28-oate

(pivaloyloxy)methyl 3β-hydroxy-30-oxolup-20(29)-en-28-oate

Example 5 Anhydride of 3β,28-diacetoxy-21,22-secolup-18-ene-21,22-dioicacid

A solution of 21,22-dioxolup-18-ene-3β,28-diyl diacetate (25 g; 45 mmol)and peroxyacetic acid (0.6 l; 32%) in chloroform (0.25 l) was vigorouslystirred until the reaction was completed (TLC was developed inhexane/ether). The colourless reaction mixture was diluted with coldwater and extracted with chloroform (3×200 ml). The combined organicphases were washed with 5% aqueous solution of potassium iodide (400ml), saturated aqueous solution of sodium sulphite (200 ml) and brine(2×200 ml), dried and evaporated. The resulting pale-yellow oil wascrystallized from chloroform/methanol to give 20.7 g (80.5%) of thetitle compound as small white crystals, m.p. 306–309° C., [α]_(D)+88° (c0.45; CHCl₃). Another way to the title compound is described in SejbalJ., Kilnot J., Bude{hacek over (s)}ínský M., Protiva J.: Collect. Czech.Chem. Commun. 56, 2936 (1991).

The ¹H NMR spectrum of the title compound is as follows: 0.85 s, 0.85 s,0.90 s, 0.91 s, 1.11 s, 1.14 d (J=7), 1.31 d (J=7), 7×3H (7×CH₃); 2.01s, 3 H, 2.05 s, 3 H (2×OAc); 2.53 dt, 1 H (J′=14.4, J″=J′″=3.5); 2.72dd, 1 H (J′3.1, J″=12.3 ); 3.26 sept, 1 H (H-20, J=7); 3.90 d, 1 H, 4.54d, 1 H (2×H-28, J=11.0); 4.47 m, 1 H (H-3α).

The following compounds were prepared by the above-mentioned procedure:

3β,28-diacetoxy-18-oxo-18,19-seco-19,20,29,30-tetranorlupan-21-oic acid[{hacek over (S)}arek J., Klinot J., Klinotová E., Sejbal J.: Chem.Listy 91, 1005 (1997)],

3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oic acid.

Example 6 3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oicacid

The anhydride of 3β,28-diacetoxy-21,22-secolup-18-ene-21,22-dioic acid(20 g; 35.1 mmol) in ethyl acetate (1 l) was added to a mixture ofruthenium dioxide (400 mg; 4 mmol), sodium metaperiodate (60 g; 280.3mmol), water (200 ml) and trifluoroacetic acid (20 ml), and the mixturewas vigorously stirred. After the reaction was completed, ethanol wasadded, the mixture was filtered, and the organic layer was filteredthrough a short silica gel column. The column was then washed with ethylacetate, the filtrate was evaporated under the reduced pressure andresidue was washed with ether and crystallized from a mixture ofdichloromethane/ether. The yield of the title compound was 10.6 g (61%),m.p. 137–140° C., [α]_(D)+40° (c 0.37; CHCl₃). [{hacek over (S)}arek J.,Klinot J., Klinotová E., Sejbal J.: Chem. Listy 91, 1005 (1997)].

The ¹³C NMR spectrum of the title compound is as follows:

213.3, 174.2, 171.1, 170.5, 80.7, 65.4, 58.8, 55.4, 50.6, 50.1, 46.8,41.1, 38.5, 37.8, 37.1, 33.9, 28.4, 27.9, 26.8, 23.5, 21.8, 21.2, 20.6,19.6, 18.1, 16.7, 16.5, 16.2, 16.0.

The following compounds were prepared by the above-mentioned procedure:

18,19,21-trioxo-18,19-secolupane-3β,28-diyl diacetate [{hacek over(S)}arek J., Klinot J., Klinotová E., Sejbal J.: Chem. Listy 91, 1005(1997)].

18,19,21,22-tetraoxo-18,19-secolupane-3β,28-diyl diacetate3β-acetoxy-21-oxolup-18-en-28-oic acid

Example 7 3β,28-diacetoxy-18-hydroxy-19,20,21,29,30-pentanorlupan-22-oicacid

To a solution of3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupane-22-oic acid (10 g;19.8 mmol) in 500 ml TEF, a solution of cerium chloride (200 ml; 1 M)and sodium borohydride (2 g; 53 mmol ) was added. The reaction mixturewas stirred for one hour. The mixture was poured into an excess of 1%aqueous solution of hydrochloric acid and extracted with chloroform(3×300 ml). The usual working up procedure gave 8.7 g (86.7%) of titlecompound, m.p. 156–159° C., [α]_(D)+48° (c 0.32; CHCl₃).

The ¹³C NMR spectrum of the title compound is as follows:

179.5, 171.1, 170.9, 80.9, 73.5, 64.5, 55.4, 51.7, 50.6, 41.0, 40.3,38.4, 37.8, 37.3, 37.1, 32.6, 27.9, 25.9 (2 C), 23.6, 21.3, 20.9, 20.8,19.9, 18.0, 16.5, 16.3, 16.1,16.0.

Example 8 (pivaloliox)methyl-3β,28-diacetoxy-18-hydroxy-19,20,21,29,30-pentanorlupan-22-oate

DBU (0.3 g; 2 mmol) and chloromethylpivalate (0.3 g; 2 mmol) were addedto the solution of3β,28-diacetoxy-18-hydroxy-19,20,21,29,30-pentanorlupan-22-oic acid (1g; 2 mmol) in a mixture of dichloromethane (5 ml) and acetonitrile (2ml). The mixture was vigorously stirred for 3 hours and then dilutedwith an ice-cold water and extracted with chloroform (3×, 10 ml).Collected organic extracts were washed with cold brine, dried andchloroform was evaporated in vacuum. The resulting viscous pale yellowoil (1.5 g) was chromatographed on silicagel, eluting with toluene.After the crystallization from methanol, 0.6 g (48%) of the titlecompound was obtained in the form of colorless needles, m.p. 235–240°C., [α]_(D)+53° (c 0.23; CHCl₃).

The ¹³C NMR spectrum obtained for the title compound is as follows:

177.4, 172.9, 171.0, 170.6, 80.8, 80.0, 73.5, 64.1, 55.4, 52.1, 50.6,41.0, 40.3, 38.8, 38.5, 37.8, 37.3, 37.1, 32.7, 27.9, 26.8 (3 C), 25.9,25.8, 23.6, 21.3, 20.9, 20.7, 19.8, 18.1, 16.5, 16.3, 16.1, 16.0.

The following esters were prepared via this general procedure:

(pivaloyloxy)methyl-3β,28-diacetoxy-18-oxo-18,19-seco-19,20,29,30-tetranorlupan-21-oate

(pivaloyloxy)methyl-3β-hydroxy-30-oxo-lup-20(29)-en-28-oate

(pivaloyloxy)methyl-3β-acetoxy-21-oxo-lup-18-en-28-oate

In the same manner, using AcM-Br instead of POM-Cl, following esterswere prepared:

Acetoxymethyl-3β,28-diacetoxy-18-hydroxy-19,20,21,29,30-pentanorlupan-22-oate

Acetoxymethyl-3β,28-diacetoxy-18-oxo-18,19-seco-19,20,29,30-tetranorlupan-21-oate

Acetoxymethyl-3β-hydroxy-30-oxolup-20(29)-en-28-oate

Acetoxymethyl-3β-acetoxy-21-oxolup-18-en-28-oate

Example 9 3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-12-en-22-oicacid

Bis(tributyltin) oxide (1.9 g; 3.2 mmol) was added to a solution of(pivaloyloxy)methyl3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-12-en-22-oate (1 g;1.6 mmol) and AIBN (20 mg) in ether (50 ml). The mixture was vigorouslystirred until the reaction was completed (TLC was developed withchloroform/ethyl acetate).

Ether was then evaporated under vacuum and the product was purified bychromatography on silicagel, eluting with chloroform/ethyl acetate.After crystallization from a dichloromethane/ether mixture the titlecompound was obtained in the form of white crystalline solid (0.5 g,62%), m.p. 138–141° C., [α]_(D)+38° (c 0.25; CHCl₃).

The ¹³C NMR spectrum of the title compound is as follows:

16.0, 16.7, 16.9, 18.1, 20.8, 21.3, 23.4, 24.2, 24.4, 24.6, 25.2, 27.8,33.4, 36.8, 37.7, 38.3, 38.7, 43.9, 46.9, 55.4, 58.9, 64.3, 80.5, 138.1,140.6, 170.5, 170.9, 171.6, 196.8.

Example 10 3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-22-oylFluoride and Similar Compounds

Diethylaminosulphur trifluoride (0.5 ml; 3.25 mmol) was added dropwiseto a solution of3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlup-22-oic acid (0.5 g;1.0 mmol) in dry chloroform (5 ml) and the reaction mixture was stirredovernight at room temperature. After the reaction was complete, themixture was slowly poured into cold water (50 ml) and extracted twicewith chloroform. The combined organic fractions were worked up in thegeneral manner and chromatographed on silica gel (20% ethyl acetate inhexane). The residue was crystalized from isopropyl alcohol to give 0.19g (38%) of the title compound as white crystals, m.p. 150–155° C.(decomp.), [α]_(D)+21° (c 0.55; CHCl₃).

The ¹³C NMR spectrum of the title compound is as follows:

211.3 s, 160.2 d (J=374), 170.1 s, 169.5 s, 80.6 s, 65.4 s, 57.5 d(J=39), 55.1 s, 50.6 s, 49.9 s, 46.8 s, 40.1 s, 38.5 s, 37.8 s, 36.1 s,33.3 s, 28.4 s, 27.9 s, 26.8 s, 23.5 s, 21.3 s, 21.2 s, 20.6 s, 19.6 s,18.0 s, 16.5 s, 16.4 s, 16.2 s, 15.9 s.

Example 11

Biological Activity of Betulinines

11.1. In Vitro Cytotoxic Activity of Betulinines on Tumor Cell Lines

One of the parameters used as the basis for colorimetric assays is themetabolic activity of viable cells. For example, a microtiter assaywhich uses the tetrazolium salt MTT is now widely used to quantitatecell proliferation and cytotoxicity [Hajdúch M, Mihál V, Minarík J,Fáber E, {hacek over (S)}afárová M, Weigl E, Antálek P.: Cytotechnology,1996, 19, 243–245]. For instance, this assay is used in drug screeningprograms and in chemosensitivity testing. Because tetrazolium salts arecleaved only by metabolically active cells, these assays exclusivelydetect viable cells. In the case of the MTT assay, yellow solubletetrazolium salt is reduced to a coloured water-insoluble formazan salt.After it is solubilized, the formazan formed can easily and rapidly bequantified in a conventional ELISA plate reader at 570 nm (maximumabsorbancy). The quantity of reduced formazan corresponds to the numberof vital cells in the culture.

Human T-lymphoblastic leukaemia cell line CEM was used for routinescreening of these compounds. To prove a common mechanism of action,selected compounds which showed activity in a screening assay weretested in a panel of cell lines (Table 2). These lines were fromdifferent species and of different histogenetic origin and they possessvarious alterations in cell cycle regulatory proteins and hormonedependence status (Table 2). The cells were maintained in Nunc/Corning80 cm² plastic tissue culture flasks and cultured in cell culture medium(DMEM with 5 g/l glucose, 2 mM glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin, 10% foetal calf serum and sodium bicarbonate). Individualcompounds were dissolved in 10% diiethylsulfoxide/saline, pH 8.0.

The cell suspensions that were prepared and diluted according to theparticular cell type and the expected target cell density (2,500–30,000cells per well based on cell growth characteristics) were added bypipette (80 μl) into 96/well microtiter plates. Inoculates were alloweda pre-incubation period of 24 hours at 37° C. and 5% CO₂ forstabilisation. Four-fold dilutions of the intended test concentrationwere added at time zero in 20 μl aliquots to the microtiter plate wells.Usually, test compounds were evaluated at six 4-fold dilutions. Inroutine testing, the highest well concentration was 250 μM, but it maydiffer, depending on the agent. All drug concentrations were examined induplicate. Incubations of cells with the test compounds lasted for 72hours at 37° C., in 5% CO₂ atmosphere and 100% humidity. At the end ofthe incubation period, the cells were assayed by using the MTT assay.Ten microliters of the MIT stock solution were pipetted into each welland incubated further for 1–4 hours. After this incubation period,formazan was solubilized by the addition of 100 μl/well of 10% SDS inwater (pH=5.5) followed by further incubation at 37° C. overnight Theoptical density (OD) was measured at 540 nm with the Labsystem iEMSReader MF(UK). The tumour cell survival (TCS) was calculated using thefollowing equitation: TCS=(OD_(drug exposed well)/meanOD_(control wells))×100%. The TCS₅₀ value, the drug concentration lethalto 50% of the tumour cells, was calculated from the obtained doseresponse curves.

To evaluate the anti-cancer activity of betulinines, their cytotoxicactivity against CEM cell line was examined using the screening assay.Potent compounds were further tested against a panel of cell lines ofdifferent histogenetic and species origin (Table 2).

TABLE 2 Cytotoxic activity of selected betulinines against a panel ofdifferent (non)malignant cell lines. Compound (TCS₅₀[μM]) Betulinic CellLine Description acid I.3 I.7 I.28 I.44 I.55 I.57 B16 mouse melanoma 362.1 B16F mouse melanoma, metastatic 4.6 4.7 SW620 human colon cancer,250 1.2 metastasis U87MG human glioblastoma 250 5.1 HepG2 humanhepatocellular 3.6 1.7 carcinoma A549 human lung adenocarcinoma 236 1.0MCF-7 human breast cancer, estrogen 194 2.3 dependent, p53+/+, Rb+/+U2OS human osteosarcoma, p53+/−, 250 1.5 Rb+/− Saos2 humanrhabdomyosarcoma, 250 1.8 p53−/−, Rb−/− BT549 human breast cancer, 2502.0 p53mut/mut MDA-MB-238 human breast cancer, estrogen 195 1.4independent, p53mut/mut DU145 human prostate cancer, 241 0.8 androgenindependent, Rb−/− HT-29 human colon cancer 250 1.6 OVCAR-3 humanovarian cancer 164 1.0 Caco-2 human colon cancer 20 3.0 MEL-3 humanmelanoma 2.7 1.3 Lymphocytes human normal lymphocytes 250 13 NIH3T3mouse immortalised fibroblasts 250 7.2 K562 human promyelocytic leukemia250 0.2 K562-CdA human promyelocytic leukemia, 250 0.3 cladrubinresistant K562-GEM human promyelocytic leukemia, 101 0.9 gemcitabinresistant K562-ARA-C human promyelocytic leukemia, 250 0.6 cytarabinresistant K562-FLUD human promyelocytic leukemia, 250 0.4 fludarabinresistant CEM human T-lymphoblastic 250 1.0 5.0 18 11 26 0.2 leukemiaCEM-DNR human T-lymphoblastic 250 0.6 1/C2 leukemia, daunorubicinresistant CEM-DNR human T-lymphoblastic 250 1.1 bulk leukemia,daunorubicin resistant CEM-VCR human T-lymphoblastic 19 3.3 1/F3leukemia, vincristin resistant CEM-VCR human T-lymphoblastic 24 2.9 3/D5leukemia, vincristin resistant CEM-VCR human T-lymphoblastic 69 2.5 bulkleukemia, vincristin resistant

In contrast to betulinic acid, which is reported to be an agentselective for neuroectodermal derived tumours, there was no significantdifference in sensitivity of betulinines to tumours of differenthistogenetic origin.

The compounds are effective in submicromolar or low micromolarconcentrations. However, the non-malignant cells, e.g. NIH3T3fibroblasts and normal human lymphocytes, tolerated substantially higherdoses of betulinines than the tumour cells suggesting a favourabletherapeutic index.

Notably, the effectiveness of betulinines was found to be identical incell lines bearing various mutations or deletions in cell cycleassociated proteins (Table 2). This indicates that these substancesshould be equally effective in tumours with various alterations oftumour suppresser genes, namely p53, Rb, etc.

Furthermore, betulinines were shown to be equally effective in drugresistant cell lines as on their maternal counterparts, therebysuggesting that classical mechanisms of multidrug resistance apparentlydo not apply to these compounds. This particular characteristic shouldbe of significant therapeutic benefit to chemotherapy resistant cancerpatients.

Finally, the cytotoxic activity of betulinines is independent of thehormonal status of cancer cells, so the compounds should be equallyeffective in treatment of hormone dependent and independent cancers.

11.2. Betulinines Induce Apoptosis in Tumour Cells.

To analyse the mechanisms of betulinine-induced cytotoxicity, it isimportant to distinguish apoptosis from the other major form of celldeath, necrosis. Finally, at the tissue level, apoptosis produces littleor no inflammation, since shrunken portions of the cell are engulfed bythe neighbouring cells, especially macrophages, rather than beingreleased into the extracellular fluid. In contrast, in necrosis,cellular contents are released into the extracellular fluid, and thushave an irritant affect on the nearby cells, causing inflammation.Secondly, at the cellular level, apoptotic cells exhibit shrinkage andblebbing of the cytoplasm, preservation of structure of cellularorganelles including the mitochondria, condensation and margination ofchromatin, fragmentation of nuclei, and formation of apoptotic bodies,thought not all of these are seen in all cell types. Thirdly, at themolecular level, a number of biochemical processes play an importantrole in the induction of apoptosis. However, the majority of them arenot well understood, and they result in activation of proteases andnucleases, which finally destruct key biological macromolecules—proteinsand DNA. For the detection of apoptotic versus necrotic modes of celldeath, the morphology was assessed by scanning electron microscopy.

A549 cell line was cultured on tissue culture treated glass coverslipsin 6-well culture plates with or without 2 μM concentration of I.3 orbetulinic acid at 37° C. and 5% CO₂ for 12 hours. Following incubation,the coverslips were washed in Hank's buffered salt solution andprocessed as described below.

Cells were fixed in 2% glutaraldehyde/PBS overnight at 4° C., dried andcovered with gold under vacuum. The surface of the cells was examinedfor typical morphologic markers of apoptosis under a scanning electronmicroscope (Tesla, Czech Republic).

Initial phase contrast microscopy examination indicated that betulininesinduce typical morphological features of apoptosis in cancer cells. Thiswas later confirmed by electron microscopy (FIG. 1). Typicalmorphological criteria of apoptosis were identified in cells treatedwith betulinine I.3: cytoplasmatic blebbing, cellular fragmentation andformation of apoptotic bodies.

11.3. In Vivo Activity of Betulinine I.3.

Animal tumour systems are important models for determining the abilityof a compound to be adsorbed into the blood stream, to penetrate intothe tumour compartment and to kill fractions of proliferating or restingtumour cells at minimally toxic doses. Although subject to criticism, invitro/in vivo models have identified all of the chemotherapeutic agentseffective in current clinical practice. It is well recognised that mostcompounds are efficacious in lymphomas and leukemias, whereas few haveproved to be effective in solid tumours, illustrating that there aremajor differences between animal models and clinical situations. Solidtumours of different histogenetic origin are implanted subcutaneously orintramuscularly into animals which are then treated with a single agent.Inhibition of solid tumour growth is thus a parameter related to theactivity of the drug. Conversely, in patients primary tumours arecontrolled by surgery and radiation, together with polychemotherapy.Usually, the acceptable criterion for activity is >50% tumourregression. However, drugs active in several different screening systemsare more likely to be effective in humans; for instance, doxorubicin,cyclophosphamide and cisplatin have a broad spectrum of activity inanimal tumour models. Moreover, with regard to toxicity and total drugexposure, a correlation has been found between nice and man for mostactive antitumor drugs.

Betulinic acid was reported to exhibit prominent activity inneuroectodermal tumours, e.g. melanoma, primitive neuroectodermaltumours (PNET) and neuroblastoma. The latter is the most lethal solidtumour of childhood as it is considered to be one of the most drugresistant tumours. In the light of this knowledge, the activity of I.3in xenotransplanted human neuroblastoma was of particular interest,although this compound demonstrated broad anti-cancer activity under invitro conditions.

Human neuroblastoma SK-N-AS cell line was obtained from ATTC. The linewas cultured in Dulbeco's modified essential medium with 4.5 gdextrose/l, 10% of foetal calf serum, 2 mM glutamine, 100 U/mlpenicillin and 100 mg/ml streptomycin. For transplantation purposes,only the cells in log phase were used.

CD-1/Ctrl-1 nu/nu nude female mice, 8 weeks old, 18–22 g weight wereused in this study (IffaCredo, France). Animals were inoculated with5.10⁶ tumour cells subcutaneously into right inquinal region. Followingtransplantation, tumor growth was measured in both the control/vehicleand the treated groups using calipers. Tumor volume (TV) was calculatedas follows: TV=(a²×b)/2, where a is width and b is length Thestatistical significance was evaluated using a non-parametric t-test.

Cisplatin (Platidiam 10 inj, sicc., Lachema, Czech Republic) was used asa control drug in this experiment. It was diluted in apyrogenic waterand applied to animals in a final volume of 0.18 ml subcutaneously atday 1.

I.3 was synthesised as described above. It was suspended at 10 mg/ml in5% dextrose and pH was adjusted to 8.0 with sodium hydroxide.

vehicle: 5% dextrose with pH adjusted to 8.0 with sodium hydroxide.

Animals were treated with drugs/vehicle 24 hours after transplantationThe following groups (with 10 animals per group) were created:

control—untreated animals

vehicle—0.2 ml of vehicle applied to animals at 3×D1,2,5,6,9,10intraperitoneally

cisplatin 4 mg/kg—0.18 ml of cisplatin solution applied to animalssubcutaneously at 1×D1

I.3 1 mg/mouse s.c.—0.1 ml of I.3 suspension applied to animals at3×D1,2,5,6,9,10 subcutaneously

I.3 2 mg/mouse s.c.—0.2 ml of I.3 suspension applied to animals at3×D1,2,5,6,9,10 subcutaneously

I.3 1 mg/mouse i.p.—0.1 ml of I.3 suspension applied to animals at3×D1,2,5,6,9,10 intraperitoneally

I.3 2 mg/mouse i.p.—0.2 ml of I.3 suspension applied to animals at3×D1,2,5,6,9,10 intraperitoneally

The results of the study are summarised in FIG. 2. The anticanceractivity of I.3 is clearly demonstrated, since there is no tumor growthin I.3 treated animals. It was statistically significant in all I.3applied groups starting from week 2 of the whole experiment. The effectwas independent of the application route (intraperitoneal orsubcutaneous) and of the dose in this application.

In contrast to I.3, cisplatin was ineffective in the management ofSK-N-AS neuroblastoma and showed no significant activity.

The anticancer activity of I.3 as a typical representative of thisgeneration of betulinines was demonstrated under in vivo conditions.This novel compound seems to be highly active against humanneuroblastoma under in vivo conditions. Together with its broadanticancer activity and novel, previously unidentified mechanism ofaction, the compounds of the present invention are believed to be oftherapeutic potential for cancer patients in the future.

11.4. Betulinine I.3 Induces Rapid Dephosphorylation of Rb Protein andApotosis Related Caspase Activation.

As discussed earlier, the G1/S transition is tightly regulated byphosphorylation of retinoblastoma protein (Rb).

Since Rb protein contains multiple phosphorylation sites for CDKs, itsphosphorylated form has molecular weight about 110 kDa, while themolecular weight of hypophosphorylated protein is only 105 kDa Thissmall difference in molecular weight is enough to separate both forms byconventional SDS-PAGE electrophoresis.

CEM cells were cultured in Dulbeco's modified essential medium with 4.5g dextrose/l, 10% of foetal calf serum, 2 mM glutamine, 100 U/mlpenicillin and 100 μg/ml streptomycin with/without below indicatedconcentrations of betulkic acid (BA) or I.3. At selected time points,cells were harvested, washed in ice cold Hank's balanced salt solutionand solubilized on ice using the SDS-PAGE sample buffer containingprotease and phosphatase inhibitors (10 μg/ml of leupeptin, 10 μg/ml ofaprotinin, 10 μg/ml of soybean trypsin inhibitor, 100 μmol of benzamide,1 mM of sodium vanadate, 1 mM of NaF, 1 mM of phenylphosphate) andboiled immediately.

Total cellular proteins (100 μg/well) were separated on SDS-PAGEelectrophoresis, blotted on polyvinyldifluoride membranes and total Rbprotein, including proteolytic fragment(s) were detected using a pRbmonoclonal antibody (Oncogene, Germany, Rb(AS5), Cat# OP66 Rev 2 Sep.1996 EB, Clone LM95.1) and visualized by chemilumniuscence (ECL-WestegBlotting System, Amersham). Details of the Western blot technique aredescribed in Ausubel, F. M., Brent, R., Kingston, P E., Moore, D. D.,Seidman, J. G., Smith, J. A., Struhl, K (Eds): Short Protocols-inMolecular Biology, 2nd edition, John Wiley & Sons, New York, Chichester,Brisbane, Toronto, Singapore, 1992, page 10–33–10–35.

The results of this study indicate that in CEM lymphoblastic leukaemiacells, Rb protein is rapidly dephosphorylated following treatment withI.3, but not betulinic acid (FIG. 3). This is demonstrated by a shift ofRb protein mass from the hyperphosphorylated form with a molecular massof about 110 kDa to the hypophosphorylated form (105 kDa). The effect ofI.3 is time and concentration dependent; at 20 μM concentration, thehypocoincident form of Rb appears as early as 15 minutes after thetreatment, while at 7 μM the same effect appears after 2 hours.Interestingly, cleavage of the Rb protein after hypophosphorylation wasaccompanied with the disappearance of a 105 kDa Rb and the appearance ofan immunoreactive fragment of Rb protein with molecular weight about. 42kDa. According to the literature, proteolytic decomposition of Rb is atypical hallmark of apoptosis due to activation of cellular caspases.

The results of this study clearly indicate that the betulinines of thepresent invention, but not betilinic acid itself are capable of inducingthe rapid dephosphorylation of key cell cycle regulatory protein Rb.This is followed by the induction of apoptosis, which has been reportedto activate cellular caspases. Activated caspases have the capacity tocleave target proteins, including Rb. This is illustrated by the timeand concentration dependent appearance of an immunoreactive fragment ofRb protein with molecular weight 42 kDa in I.3 treated cells.

11.5. Betulinine I.3 Induces G1 Block and Apoptosis in Tumour Cells.

Hypophosphorylation of Rb protein is accompanied with cell cycle blockon the G1/S transition. To investigate whether incubation of tumourcells with betulinines will result in cell cycle block and/or apoptosis,a flow-cytometry study was performed with measurement of total DNAcontent in CEM cells treated with/without I.3. Taxol (paclitaxel) wasused as a positive control, since this drug is known to result in thecumulation of cells in the G2 phase.

Briefly, CEM cells were cultured in Dulbeco's modified essential mediumwith 4.5 g dextrose/l, 10% of foetal calf serum, 2 mM glutamine, 100U/ml penicillin and 100 μg/ml streptomycin with/without the indicatedconcentrations of paclitaxel or I.3 (FIG. 4A). At selected time points,cells were harvested, washed in ice cold Hank's balanced salt solution(HBSS) and fixed in 70% ethanol at −20° C. overnight. The next day,ethanol was removed by centrifugation, cells were washed twice in HBSSand a cellular pellet (10⁶ cells) was reconstituted in staining solution(propidium iodide 60 μg/ml, DNA-se free RNA-se 175 U/ml in HBSS) for 15minutes at 37° C. The DNA content of individual cells was analyzed onflow cytometer Excalibur (Becton and Dickinson) at excitation wavelengthof 488 nm.

As indicated in FIG. 4A,B I.3 induces cumulation of cells in G0/G1phases of the cell cycle, which is accompanied by rapid apoptosis(appearance of sub-G1 cells). The effect is concentration and timedependent. Induction of G1 block and apoptosis is consistent withdephosphorylation of Rb protein

11.6. Betulinine I.3 Induces Rapid Histone Acetylation.

Cellular cytotoxicity studies (data not shown) demonstrated that thecytotoxic activity of I.3 strongly correlates with actinomycin D(p<0.00001). Since actinomycin D is a well known inhibitor of RNApolymerases, these results suggest that I.3 is targeting transcriptionalcomplexes.

As discussed earlier, the G1/S transition is tightly regulated byphosphorylation of retinoblastoma protein (Rb). Hypophosphorylated Rbsilences specific genes that are active in the S phase of the cell cycleand which are regulated by E2F transcription factors. Rb binds to theactive domain of E2F and then actively repress the promoter by amechanism that is poorly understood. Recent studies show that Rbassociates with a histone deacetylase (HDAC 1 and 2) through the Rbpocket domain. Rb recruits histone deacetylase to E2F and thus Rbcooperates with HDAC to repress the E2F-regulated promotor of the gene.Inhibition of histone deacetylase activity by specific inhibitortrichostatin A (TSA) inhibits Rb-mediated repression of a E2Ftranscriptional activity.

In order to provide the evidence of interference of betulinines withtranscription, the ability of I.3 to modify histone acetylation wasinvestigated.

CEM cells were cultured in Dulbeco's modified essential medium with 4.5g dextrose/l, 10% of foetal calf serum, 2 mM glutamine, 100 U/mlpenicillin and 100 μg/ml streptomycin with/without the indicatedconcentrations of HDAC inhibitor TSA or I.3. At selected time points,cells were harvested, washed in ice cold Hank's balanced salt solutionand solubilized on ice using SDS-PAGE sample buffer containing proteaseand phosphatase inhibitors (10 μg/ml of leupeptin, 10 μg/ml ofaprotinin, 10 μg/ml of soybean trypsin inhibitor, 100 μmol of benzamide,1 mM of sodium vanadate, 1 mM of NaF, 1 mM of phenylphosphate) andboiled immediately.

Total cellular proteins (100 μg/well) were separated on SDS-PAGEelectrophoresis, blotted on polyvinyldifluoride membranes and acetylatedhistone was detected using a anti-acetyl(Lys9)-histone H3 rabbitpolyclonal antibody (Cell Signalling Technology, Beverly, Mass.; Cat#9671L) and visualized by chemilumniuscence (ECL-Western Blotting System,Amersham). Details of the Western blot technique are described inAusubel, F. M., Brent, R, Kingston, R. E., Moore, D. D., Seidman, J. G.,Smith, J. A., Struhl, K. (Eds): Short Protocols in Molecular. Biology,2nd edition, John Wiley & Sons, New York, Chichester, Brisbane, Toronto,Singapore, 1992, page 10–33–10–35.

The results of this study indicate that in CEM lymphoblastic leukaemiacells, H3 histone is rapidly acetylated in Lys9 position following thetreatment of both TSA and betulinine I.3 (FIG. 5). The effect of I.3 istime and concentration dependent; at 20 μM (10×IC₅₀) acetylated histoneappears as soon as 30 minutes after incubation. Interestingly, CEM cellscultured with 2 μM of I.3 (IC₅₀) show histone acetylation in time pointsin between 45–120 minutes, which is consistent with degradationhalf-life of I.3 under in vitro conditions (data not shown). On theother hand, however, TSA at 0.24 μM (IC₅₀) showed no significant abilityto increase histone acetylation, suggesting that mechanism(s) ofcytotoxic activity of TSA may be more complex tat we originallyrealised.

The results of this study clearly indicate that the betulinines of thepresent invention, are capable of inducing the rapid histoneacetylation. It is generally accepted that N termini of core histonesare central to the processes that modulate nucleosome structure.Hyperacetylation of the histones reduces their ability to constrain thepath of DNA within chromatin, resulting in allosteric changes innucleosomal conformation, destabilization of internucleosomal contacts,and an increase in the accessability of nucleosomal DNA to transcriptionfactors. The amount of histone acetylation is determined by anequilibrium between histone acetyltransferases (HAT) and histonedeacetylases (HDAC). The balance plays an important role in theregulation of gene transcription as well as the genesis or suppressionof cancer. Unfortunately, the question of whether betulinines inhibitHDAC directly or indirectly remains open. As discussed earlier,betulinines induce rapid dephosphorylation of Rb protein.Hypophosphorylated Rb was shown to bind both HDAC and E2F-DP1 complex.It has been recently reported that E2F-1, -2, and -3, but not E2F-4, -5,and -6, associate with and are acetylated by p300 and cAMP-responseelement-binding protein acetyltransferases. Acetylation occurs at threeconserved lysine residues located at the N-terminal boundary of theirDNA binding domains. Acetylation of E2F-1 in vitro and in vivo markedlyincreases its binding affinity for a consensus E2F DNA-binding site,which is paralleled by enhanced transactivation of an E2F-responsivepromoter. Acetylation of E2F-1 was reversed by HDAC1, indicating thatreversible acetylation is a mechanism for regulation also of non-histoneproteins. Thus, inhibition of HDAC by synthetic compounds should resultin both histone and transcription factor acetylation resulting intranscriptional activation of responsive genes. Interestingly, a numberof oncosupressors and virus promotor driven genes are transcritionallyrepressed in cancer/transfected/virus infected cells due to constitutivedeacetylation of specific promoters. Inhibition of HDAC results inre-activation of onco-suppressors and genes under virus promotor. Thoseskilled in the art will recognise that betulinines could be used notonly in treatment of cancer, but also in the treatment of diseases or intherapeutic approaches, where transcriptional repression of specificgene(s) is undesired, for instance transcriptional silencing of vectorsused in gene therapy.

Notably, since these potential drugs do not exhibit any CDK inhibitoryactivity (data not shown), we expect that they possess a new, previouslyunreported mechanism of action.

Those skilled in the art will recognise, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the claims.

1. A compound of the formula Ia, or a pharmaceutically acceptable saltthereof;

wherein: X¹ is C═O, or CHOR^(1a); X³is C═O, CHOR^(1b), or CHOCOR^(1b);R¹ and R³ are each independently H or lower alkyl; R², R⁴ and R⁵ areeach independently lower alkyl; R⁶ is H or absent if “a” is a doublebond; R⁷ is H, COOR^(1c), YOCOR^(1c), COOYOCOR^(1e), YCOOR^(1e); R⁸ isH, COOR^(1d), YCOOR^(1d), YCOOR¹⁰, YCOH^(al), COOYOCOR^(1d), CH₂OR^(1d),CH₂COCOR^(1d), COCOCOR^(1d), or R⁷ and R⁸ together are ═CH₂; R¹⁰ islower alkyl substituted Hal; “a” is a double bond or single bond; andwherein Y═(CH₂)_(n) n=0 to 5; R^(1a-1e) are the same or different groupsof R¹ with the proviso that when X¹ is CHOH, X³ is C═O, “a” is a singlebond, R¹⁻⁵ are Me, and R⁶ is H, when R⁷ is H, R⁸ is other than H,CH₂COOH or CH₂COOMe; when R⁷ is CH₂OAc, R⁸ is other than CH₂COOH; or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein: X¹ is CHOR^(1a); and R⁸ is H, COOR^(1d), YCOOR^(1d),COOYOCOR^(1d), CH2OR^(1d), CH₂COCOR^(1d), or COCOCOR^(1d).
 3. Thecompound of claim 1, wherein: R²⁻⁵ are all methyl and R¹ is as definedbelow for the relevant group R^(1a-1d); X¹ is —CHOR^(1a), wherein R^(1a)is H; X³ is C═O, CHOH or CHOAc; R⁷ H, COOH, COOMe, CH₂OAc,COOYOCOR^(1e), YCOOR^(1e) where Y is CH₂ and R^(1e) is C¹⁻⁴ alkyl; R⁸ is—COOR^(1d), R^(1d) is H or methyl; —YCOOR^(1d), wherein Y is CH₂ andR^(1d) is H, methyl or ethyl; —COOYOCOR^(1d), wherein Y is CH₂ andR^(1d) is C₁₋₄ alkyl; —CH₂OR^(1d), wherein R^(1d) is C₁₋₄ alkyl;CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄ alkyl.
 4. Thecompound of claim 1 wherein “a” is a single bond and R⁶ is H.
 5. Thecompound of claim 2 wherein “a” is a single bond and R⁶ is H.
 6. Thecompound of claim 3 wherein “a” is a single bond and R⁶ is H.
 7. Acompound of formula Ia, or a pharmaceutically acceptable salt thereof

wherein the compound is selected from the group consisting of: No. X¹ R¹X³ a R^(2–5) R⁶ R⁷ R⁸ 1.4 CHOAc CH₃ C═O single Me H CH₂OAc COOMe 1.7CHOAc CH₃ C═O single Me H H CH₂OEt 1.8 CHOAc CH₃ CHOH single Me H CH₂OAcCOOH 1.9 CHOAc CH₃ C═O double Me absent CH₂OAc COOMe 1.10 CHOAc CH₃ CHOHsingle Me H R⁷ and R⁸ together: ═CH₂ 1.11 CHOAc CH₃ CHOAc single Me HCH₂OAc COOMe 1.12 CHOAc CH₃ C═O single Me H COOMe CH₂COCOPr¹ 1.13 CHOAcCH₃ C═O single Me H COOMe COOH 1.14 CHOAc CH₃ C═O single Me H COOMeCH₂COOH 1.15 CHOAc CH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) 1.16CHOAc CH₃ CHOH single Me H CH₂OAc COOMe 1.18 CHOAc CH₃ C═O single Me HCH₂OAc COOCH₂OCOBu^(t) 1.19 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOMe1.20 CHOAc CH₃ C═O double Me absent CH₂OAc COOCH₂OCOBu^(t) 1.21 CHOAcCH₃ C═O double Me absent CH₂OAc COOCH₂OCOMe 1.22 CHOAc CH₃ C═O double Meabsent CH₂OAc COOH 1.24 CHOAc CH₃ C═O single Me H R⁷ and R⁸ together:═CH₂ 1.26 CHOH H C═O single Me H CH₂OAc COOH 1.27 CHOH H C═O single Me HCH₂OAc COOMe 1.28 CHOAc CH₃ C═O double Me absent COOCH₂OCOMe CH₂COCOPr¹1.29 CHOAc CH₃ C═O single Me H COOCH₂OCOBut CH₂COCOPr¹ 1.30 CHOAc CH₃C═O single Me H from R⁷ and R⁸: ═CH₂OC(O)CH₂ 1.31 CHOAc CH₃ CHOAc singleMe H CH₂OAc COOH 1.32 CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t)1.33 CHOAc CH₃ CHOH single Me H CH₂OAc COOMe 1.34 CHOAc CH₃ CHOAc singleMe H CH₂OAc COOMe 1.35 CHOAc CH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t)1.36 CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) 1.37 CHOAc CH₃C═O single Me H CH₂OAc COOMe 1.39 CHOAc CH₃ C═O single Me H COOMeCH₂COOMe 1.40 CHOAc CH₃ C═O single Me H COOCH₂OCOBu^(t) CH₂COOMe 1.41CHOAc CH₃ C═O single Me H COOCH₂OCOMe CH₂COOMe 1.42 CHOAc CH₃ CHOHsingle Me H R⁷ and R⁸ together: ═CH₂ 1.43 CHOAc CH₃ CHOH single Me H H H1.44 CHOC CH₃ C═O single Me H CH₂OAc COOH OCH₂Cl 1.45 CHOAc CH₃ CHOHsingle Me H CH₂OAc COOH 1.46 CHOAc CH₃ CHOAc single Me H CH₂OAc COOH1.47 CHOAc CH₃ CHOAc single Me H R⁷ and R⁸ together: ═CH₂ 1.48 CHOAc CH₃CHOAc single Me H H H 1.51 CHOH CH₃ CHOH single Me H H CH₂COOMe 1.52CHOAc CH₃ CHOH single Me H H CH₂COOMe 1.53 CHOH CH₃ CHOH single Me HCH₂COOMe H 1.54 CHOAc CH₃ CHOAc single Me H CH₂COOMe H 1.57 CHOH CH₃ C═Osingle Me H CH₂OAc COOH 1.58 CHOAc CH₃ C═O single Me H CH₂OAc COF


8. A compound of the formula Ia, or a pharmaceutically acceptable saltthereof;

wherein: X¹ is C═NOR^(1a), CHOCOR^(1a), CHOCOY-Hal, CHOC(O)OR⁹,CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I, F; X³ is C═O,CHOR^(1b), or CHOCOR^(1b); R¹⁻⁵ are each independently H or lower alkyl;R⁶ is H or absent if “a” is a double bond; R⁷ is H, COOR^(1c),YOCOR^(1c), COOYOCOR^(1e), YCOOR^(1e); R⁸ is H, COOR^(1d), YCOOR^(1d),YCOOR¹⁰, YCOHal, COOYOCOR^(1d), CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d),or R⁷ and R⁸ together are ═CH₂; R⁹ is an OH-substituted alkyl group, anether group or a cyclic ether; R¹⁰ is lower alkyl substituted Hal; “a”is a double bond or single bond; and wherein Y═(CH₂)_(n) n=0 to 5;R^(1a-1e) are the same or different groups of R¹ with the proviso thatwhen X¹ is CHOAc, X³ is C═O, “a” is a single bond, R¹⁻⁵ are Me, R⁶ is H;when R⁷ is CH₂OAc, R⁸ is other than COOH, CH₂COCOPr^(i), COCOCOPr^(i) orCH₂COOH; when R⁷ is H, R⁸ is other than H, CH₂COOMe or CH₂COOH, or apharmaceutically acceptable salt thereof.
 9. The compound of claim 8,wherein: X¹ is CHOCOR^(1a), or CHOCOY-Hal; and R⁸ is H, COOR^(1d),YCOOR^(1d), COOYOCOR^(1d), CH2OR^(1d), CH₂COCOR^(1d) or COCOCOR^(1d).10. The compound of claim 8, wherein: R²⁻⁵ are all methyl and R¹ is asdefined below for the relevant group R^(1a-1d); X¹ is —CHOCOCH₂Cl; or—CHOCOR^(1a), wherein R^(1a) methyl; X³ is C═O, CHOH or CHOAc; R⁷ is H,COOH, COOMe, CH₂OAc, COOYOCOR^(1e), YCOOR^(1e) where Y is CH₂ and R^(1e)is C¹⁻⁴ alkyl; R⁸ is —COOR^(1d), R^(1d) is H or methyl; —YCOOR^(1d),wherein Y is CH₂ and R^(1d) is H, methyl or ethyl; —COOYOCOR^(1d),wherein Y is CH₂ and R^(1d) is C₁₋₄ alkyl; —CH₂OR^(1d), wherein R^(1d)is C₁₋₄ alkyl; —CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄alkyl.
 11. The compound of claim 8, wherein “a” is a single bond and R⁶is H.
 12. The compound of claim 9 wherein “a” is a single bond and R⁶ isH.
 13. The compound of claim 10 wherein “a” is a single bond and R⁶ isH.
 14. A method of treating a proliferative disease comprisingadministering a therapeutically effective amount of a compound offormula I, or a pharmaceutically acceptable salt thereof

wherein: X¹ is C═O, C═NOR^(1a), CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal,CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I, F; X³ isC═O, CHOR^(1b), or CHOCOR^(1b); R¹⁻⁵ are each independently H or loweralkyl; R⁶ is H or absent if “a” is a double bond; R⁷ is H, COOR^(1c),YOCOR^(1c), COOYOCOR^(1e), YCOOR^(1e); R⁸ is H, COOR^(1d), YCOOR^(1d),YCOOR¹⁰, YCOHal, COOYOCOR^(1d), CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d),or R⁷ and R⁸ together are ═CH₂; R⁹ is an OH-substituted alkyl group, anether group or a cyclic ether; R¹⁰ is lower alkyl substituted Hal; “a”is a double bond or single bond; and wherein Y═(CH₂)_(n) n=0 to 5;R^(1a-1e) are the same or different groups of R¹.
 15. The method ofclaim 14, wherein: X¹ is CHOR^(1a), CHOCOR^(1a) or CHOCOY-Hal; and R⁸ isH, COOR^(1d), YCOOR^(1d), COOYOCOR^(1d), CH₂OR^(1d), CH₂COCOR^(1d) orCOCOCOR^(1d).
 16. The method of claim 14, wherein: R²⁻⁵ are all methyland R¹ is as defined below for the relevant group R^(1a-1d); X¹ is—CHOCOCH₂Cl; or —CHOR^(1a) OR CHOCOR^(1a), wherein R^(1a) is H andmethyl respectively; X³ is C═O, CHOH or CHOAc; R⁷ is H, COOH, COOMe,CH₂OAc, COOYOCOR^(1e) or YCOOR^(1e) where Y is CH₂ and R^(1e) is C₁₋₄alkyl; R⁸ is —COOR^(1d), wherein R^(1d) is H or methyl; —YCOOR^(1d),wherein Y is CH₂ and R^(1d) is H, methyl or ethyl —COOYOCOR^(1d),wherein Y is CH₂ and R^(1d) is C₁₋₄ alkyl; —CH₂OR^(1d), wherein R^(1d)is C₁₋₄ alkyl; CH₂COCOR^(1d) or COCOCOR^(1d), wherein R^(1d) is C₁₋₄alkyl.
 17. The method of claim 14 wherein “a” is a single bond and R⁶ isH.
 18. The method of claim 15 wherein “a” is a single bond and R⁶ is H.19. The method of claim 16 wherein “a” is a single bond and R⁶ is H. 20.A method of treating a proliferative disease comprising administering acompound of formula I, or a pharmaceutically acceptable salt thereof,

wherein the compound is selected from the group consisting of: No. X¹ R¹X³ a R^(2–5) R⁶ R⁷ R⁸ 1.1 CHOAc CH₃ C═O single Me H CH₂OAc CH₂COCOPr¹1.2 CHOAc CH₃ C═O single Me H CH₂OAc CH₂COCOPr¹ 1.3 CHOAc CH₃ C═O singleMe H CH₂OAc COOH 1.4 CHOAc CH₃ C═O single Me H CH₂OAc COOMe 1.5 CHOAcCH₃ C═O single Me H H H 1.6 CHOAc CH₃ C═O single Me H H CH₂COOH 1.7CHOAc CH₃ C═O single Me H H CH₂OEt 1.8 CHOAc CH₃ CHOH single Me H CH₂OAcCOOH 1.9 CHOAc CH₃ C═O double Me absent CH₂OAc COOMe 1.10 CHOAc CH₃ CHOHsingle Me H R⁷ and R⁸ together: ═CH₂ 1.11 CHOAc CH₃ CHOAc single Me HCH₂OAc COOMe 1.12 CHOAc CH₃ C═O single Me H COOMe CH₂COCOPr¹ 1.13 CHOAcCH₃ C═O single Me H COOMe COOH 1.14 CHOAc CH₃ C═O single Me H COOMeCH₂COOH 1.15 CHOAc CH₃ CHOH single Me H CH₂OAc COOCH₂OCOBu^(t) 1.16CHOAc CH₃ CHOH single Me H CH₂OAc COOMe 1.17 CHOAc CH₃ C═O single Me HCH₂OAc CH₂COOH 1.18 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOBu^(t)1.19 CHOAc CH₃ C═O single Me H CH₂OAc COOCH₂OCOMe 1.20 CHOAc CH₃ C═Odouble Me absent CH₂OAc COOCH₂OCOBu^(t) 1.21 CHOAc CH₃ C═O double Meabsent CH₂OAc COOCH₂OCOMe 1.22 CHOAc CH₃ C═O double Me absent CH₂OAcCOOH 1.24 CHOAc CH₃ C═O single Me H R⁷ and R⁸ together ═ CH₂ 1.26 CHOH HC═O single Me H CH₂OAc COOH 1.27 CHOH H C═O double Me absent CH₂OAcCOOMe 1.28 CHOAc CH₃ C═O single Me H COOCH₂OCOMe CH₂COCOPr¹ 1.29 CHOAcCH₃ C═O single Me H COOCH₂OCOBut CH₂COCOPr¹ 1.30 CHOAc CH₃ C═O single MeH from R⁷ and R⁸: CH₂OC(O)CH₂ 1.31 CHOAc CH₃ CHOAc single Me H CH₂OAcCOOH 1.32 CHOAc CH₃ CHOAc single Me H CH₂OAc COOCH₂OCOBu^(t) 1.33 CHOAcCH₃ CHOH single Me H CH₂OAc COOMe 1.34 CHOAc CH₃ CHOAc single Me HCH₂OAc COOMe 1.35 CHOAc CH₃ CHOH single Me H CH₂Oac COOCH₂OCOBu^(t) 1.36CHOAc CH₃ CHOAc single Me H CH₂Oac COOCH₂OCOBu^(t) 1.37 CHOAc CH₃ C═Osingle Me H CH₂Oac COOMe 1.38 CHOAc CH₃ C═O single Me H H CH₂COOMe 1.39CHOAc CH₃ C═O single Me H COOMe CH₂COOMe 1.40 CHOAc CH₃ C═O single Me HCOOCH₂OCOBu^(t) CH₂COOMe 1.41 CHOAc CH₃ C═O single Me H COOCH₂OCOMeCH₂COOMe 1.42 CHOAc CH₃ CHOH single Me H R⁷ and R⁸ together: ═CH₂ 1.43CHOAc CH₃ CHOH single Me H H H 1.44 CHOCOCH₂Cl CH₃ C═O single Me HCH₂OAc COOH 1.45 CHOAc CH₃ CHOH single Me H CH₂OAc COOH 1.46 CHOAc CH₃CHOAc single Me H CH₂OAc COOH 1.47 CHOAc CH₃ CHOAc single Me H R⁷ and R⁸together: ═CH₂ 1.48 CHOAc CH₃ CHOAc single Me H H H 1.49 CHOH CH₃ C═Osingle Me H H CH₂COOH 1.50 CHOH CH₃ C═O single Me H H CH₂COOMe 1.51 CHOHCH₃ CHOH single Me H H CH₂COOMe 1.52 CHOAc CH₃ CHOH single Me H HCH₂COOMe 1.53 CHOH CH₃ CHOH single Me H CH₂COOMe H 1.54 CHOAc CH₃ CHOAcsingle Me H CH₂COOMe H

1.57 CHOH CH₃ C═O single Me H CH₂OAc COOH 1.58 CHOAc CH₃ C═O single Me HCH₂OAc COF


21. A method of treating a proliferative disease comprisingadministering a therapeutically effective amount of a compound selectedfrom the group consisting of:


22. A method of preparing a pharmaceutical composition comprisingadmixing a compound of formula I and a pharmaceutical carrier, excipientor diluent, wherein said compound of formula I is:

wherein: X¹ is C═O, C═NOR^(1a), CHOR^(1a), CHOCOR^(1a), CHOCOY-Hal,CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, and Hal is Br, Cl, I, F: X³ isC═O, CHOR^(1b), or CHOCOR^(1b); R¹⁻⁵ are each independently H or loweralkyl; R⁶ is H or absent if “a” is a double bond; R⁷ is H, COOR^(1c),YOCOR^(1c), COOYOCOR^(1e), YCOOR^(1e); R⁸ is H, COOR^(1d), YCOOR^(1d),YCOOR¹⁰, YCOHal, COOYOCOR^(1d), CH₂OR^(1d), CH₂COCOR^(1d), COCOCOR^(1d),or R⁷ and R⁸ together are ═CH₂; R⁹ is an OH-substituted alkyl group, anether group or a cyclic ether; R¹⁰ is lower alkyl substituted Hal; “a”is a double bond or single bond; and wherein Y═(CH₂)_(n) n=0 to 5;R^(1a-1e) are the same or different groups of R¹.
 23. A method ofpreparing a compound of formula I, or a pharmaceutically acceptable saltthereof,

wherein “a” is a double bond X¹ is C═O, C═NOR^(1a), CHOR^(1a),CHOCOR^(1a), CHOCOY-Hal, CHOC(O)OR⁹, CHOC(O)OR^(1a), CHOC(O)OR¹⁰, andHal is Br, Cl, I, F; X³ is C═O, CHOR^(1b), or CHOCOR^(1b); R¹⁻⁵ are eachindependently H or lower alkyl; R⁶ is H or absent if “a” is a doublebond; R⁷ is H, COOR^(1c), YOCOR^(1c), COOYOCOR^(1e), YCOOR^(1e); R⁸ isH, COOR^(1d), YCOOR^(1d), YCOOR¹⁰, YCOHal, COOYOCOR^(1d), CH₂OR^(1d),CH₂COCOR^(1d), COCOCOR^(1d), or R⁷ and R⁸ together are ═CH₂; R⁹ is anOH-substituted alkyl group, an ether group or a cyclic ether; R¹⁰ islower alkyl substituted Hal; and wherein Y═(CH₂)_(n) n=0 to 5; R^(1a-1e)are the same or different groups of R¹, the method comprising:

oxidizing a compound of formula 1b to a compound of formula 1c; (ii)reducing said compound of formula 1c to form a compound of formula 1d;and (iii) converting said formula of 1d to a compound of formula I asdefined above.
 24. The method of claim 23, wherein the compound offormula 1b is oxidized to 1c by treating sequentially with seleniumdioxide, peroxyacetic acid and ruthenium tetroxide.
 25. The method ofclaim 23, wherein the reduction of 1c to 1d is a stereoselectivereduction.
 26. The method of claim 23, wherein the reduction of 1c to 1dis a stereoselective reduction.
 27. The method of claim 23, wherein thecompound of formula 1c is reduced to 1d by treating with sodiumborohydride in the presence of cerium (III) salt.
 28. The method ofclaim 24, wherein the compound of formula 1c is reduced to 1d bytreating with sodium borohydride in the presence of cerium (III) salt.29. The method of claim 25, wherein the compound of formula 1c isreduced to 1d by treating with sodium borohydride in the presence ofcerium (III) salt.
 30. The method of claim 26, wherein the compound offormula 1c is reduced to 1d by treating with sodium borohydride in thepresence of cerium (III) salt.